We propose to develop and apply techniques, primarily based on Fourier- transform infrared spectroscopy, to investigate protein structure, dynamics, and function at the level of the individual groups. One phase of this project will involve polarized IR studies of oriented single crystals. The binding geometry of small molecules to metalloproteins will be determined and related to protein control of active site function. We will use 1R difference techniques to perform single site pH titrations and deduce conformational changes induced by active site reactions. We will identify changes in protein structural or dynamic properties introduced by crystallization through spectroscopic comparisons with solutions. A second, parallel phase of the project will use sensitive difference techniques to explore IR spectral changes during photoinduced reactions. The use of continuous, impulsive, and modulated laser excitation will allow the study of reversible and irreversible reactions that have a wide range of quantum yields. We will use these techniques to investigate structural alterations associated with protein folding, UV radiation damage, and the coupling of protein conformation to ligand binding and redox reactions at the active site. We will also initiate studies aimed at exploring the connection between hydration and protein function. The long-term objectives of this work have both specific and general aspects. On a specific level, reactions of electrons and oxygen with metalloproteins play essential roles in oxidative phosphorylation. We expect that diatomic molecules will become increasingly important for understanding intracellular processes, with the growing awareness of the diverse biological roles of nitric oxide and carbon monoxide in intracellular communication and regulation. On a more general level, the relative simplicity of the reactions in most of the proteins studied makes them ideal model systems with which-to address questions about how protein conformation controls active site reactivity, and how ligand binding alters protein conformation. Fundamental insights into such generic questions will become increasingly important both in order to understand biological systems at the molecular level and to adapt and redesign biological molecules for medical and technological applications.